Ultrastructure of Sarcocystis spp. (Protozoa: Apicomplexa) in rodents from North Sulawesi and West Java, Indonesia

Tissue cysts of the protozoan genus Sarcocystis were detected in the skeletal muscles of 16 (40%) of 40 wild rodents captured in North Sulawesi and West Java, Indonesia. Two types of cysts were found to differ in their morphological characteristics. Macroscopic and microscopic cysts bounded by thick radially-striated cyst walls were detected at both locations in a total of 13 rodents belonging to seven different species (Bunomys chrysocomus, B. fratrorum, Maxomys bartelsii, M. musschenbroekii, Paruromys dominator, Rattus xanthurus and R. exulans). The primary cyst walls contained numerous broad spatula-like protrusions and the cysts were identified as S. singaporensis Zaman and Colley, 1976. In contrast, microscopic cysts bounded by thin smooth cyst walls were detected in seven rodents belonging to three different species captured at Toraut in North Sulawesi (B. chrysocomus, B. fratrorum and P. dominator). Ultrastructural examination revealed numerous slender hair-like protrusions of their primary cyst walls. It is proposed that these cysts be named S. sulawesiensis sp. n. on the basis of their unique morphological characteristics, their intermediate host range and their limited geographic distribution. Mixed infections by both species were found in three rodent species (B. chrysocomus, B. fratrorum and P. dominator).     

Assignment of disulfide bonds in proteins by fast atom bombardment mass spectrometry

A rapid and sensitive method for assignment of disulfide bonds using fast atom bombardment mass spectrometry is described for hen egg white lysozyme and bovine ribonuclease A. The protein is initially digested to a mixture of peptides using chemical and enzymatic methods under conditions which minimize disulfide bond reduction and exchange. The digested sample is analyzed directly by fast atom bombardment mass spectrometry before and after chemical reduction of cystine residues. An important feature of the method is that it is not necessary to completely resolve the peptides in the digest chromatographically prior to analysis. The disulfide-containing peptides are also characterized directly by prolonged exposure of the sample to the high energy xenon atom beam which results in the reduction of cystine residues. Intra- as well as interchain disulfide bond assignments are made on the basis of the mass difference between the molecular ions (MH+) of the oxidized and reduced peptides. Confirmation of the mass assignments may be obtained from the mass spectra of the digests after one cycle of manual Edman degradation. Although the quantity of protein required to unambiguously assign all of the disulfide linkages will depend on the ease with which the appropriate peptide fragments can be formed, results from these studies indicate that approximately 1 nmol of protein is usually sufficient.     

Transmembrane signaling by the B subunit of cholera toxin: increased cytoplasmic free calcium in rat lymphocytes

It has previously been shown that the B subunit of cholera toxin, which binds solely to the plasma membrane ganglioside GM1, stimulates the proliferation of rat thymic lymphocytes (Spiegel, S., P. H. Fishman, and R. J. Weber, 1985, Science [Wash. DC], 230:1285-1287). The purpose of this study was to identify which transmembrane signaling system(s) are activated by the B subunit of cholera toxin. We compared the effects of B subunit and concanavalin A (Con A), a potent mitogenic lectin, on a number of second messenger systems that are putative mediators of T cell activation. Changes in the fluorescence of quin2-loaded cells revealed that mitogenic doses of either B subunit or Con A induced rapid and sustained increases in cytoplasmic free Ca2+ ([Ca2+]i). Within 5 min, [Ca2+]i increased from a basal level of 69 +/- 4 to 136 +/- 17 and 185 +/- 24 nM, respectively. The effects of B subunit and Con A were additive and largely dependent on the presence of extracellular Ca2+, though release of Ca2+ from intracellular stores could be detected for Con A, but not B subunit, using indo-1. The B subunit had no effect on either inositol phosphate levels or on the distribution of protein kinase C, indicating that, unlike Con A, the B subunit does not activate phosphoinositide hydrolysis. Fluorimetric measurements on cells loaded with bis(carboxyethyl)-5,6-carboxyfluorescein revealed that Con A induced a rapid cytoplasmic alkalinization via activation of Na+/H+ exchange, whereas B subunit had no effect on intracellular pH. Finally, by monitoring bis-oxonol fluorescence, we found that Con A induced a small hyperpolarization of the membrane potential, whereas B subunit had no acute effect. These data suggest that the biological effects of B subunit are mediated by an increase in [Ca2+]i resulting from a net influx of extracellular Ca2+.     

Studies on the metabolism of retinol-binding protein by primary hepatocytes from retinol-deficient rats

Studies were conducted to explore the regulation of retinol-binding protein (RBP) metabolism in cultured primary hepatocytes from retinol-deficient rats. Newly isolated hepatocytes from retinol-deficient rats contained elevated levels (3.4-fold) of RBP, compared to hepatocytes from normal (retinol-adequate) rats. Addition of retinol to retinol-depleted hepatocytes stimulated RBP secretion by the cells in a concentration-dependent manner. Maximal stimulation of RBP secretion was seen with a retinol level of 0.3 micrograms/ml. The effect of retinol was quite rapid, and was evident by 20 minutes after addition of retinol to the medium. Stimulation of RBP secretion was only seen during the first few hours after retinol addition. The effect of retinol was specific for RBP; thus, retinol had no effect on the secretion rates of transthyretin or albumin. Addition of retinoic acid also stimulated RBP secretion by retinol-deficient hepatocytes. Addition of dexamethasone to retinol-deficient cells did not maintain the initial rate of RBP secretion. Dexamethasone also had no effect on the secretion of transthyretin or albumin by these cells. The effects of retinol and of dexamethasone seen here with retinol-depleted cells differed dramatically from effects seen in other studies with normal (retinol-adequate) hepatocytes. Thus, with normal cells, dexamethasone maintains RBP, TTR, and albumin production and secretion rates close to initial rates. Also in normal hepatocytes, with ample retinol available within the cell, addition of exogenous retinol does not appear to influence RBP secretion. In contrast, and as shown previously in intact rats, in retinol deficiency the availability of retinol specifically regulates the secretion of RBP by hepatocytes.     

Immunostimulation of antibody-producing cells and humoral antibody to fish bacterins by a biological response modifier

Numbers of splenic antibody-producing cells and humoral antibody titres were elevated during immunization regimes in rainbow trout when the bacterins Yersinia ruckeri or Aeromonas salmonicida O-antigen preparations were mixed with the immunostimulator FK-565. Fish sampled 14 days after injection showed a marked increase in the immune response when doses of 5, 10 or 100 μg of antigen were used. The immunostimulator may aid initial antigen uptake and processing.     

Methods of hatching the eggs and rearing the fundatrices of the English grain aphid, Sitobion avenae

Several methods for hatching the eggs and rearing individuals of the first generation (fundatrices) of Sitobion avenae were investigated. The most successful methods were incubation of the eggs on grass seedlings at 2°C and rearing the fundatrices on grass seedlings (overall survival 66%) and incubation of the eggs in plastic boxes at 2°C and rearing the fundatrices on wheat seedlings (overall survival 62%).

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Résumé L'éclosion des oeufs de S. avenae peut être induite par le transfert à 10°C ou 12°C, après une incubation de 75–120 jours à 2°C. Le pourcentage le plus élevé d'éclosions a été obtenu quand les oeufs avaient incubé pendant 100 à 110 jours à 2°C (67% at 71.5% respectivement) dans des petites boîtes de plastique, ou pendant 100 jours à 2°C sur des pousses de graminées (73.5%). Si les oeufs sont pondus sur blé, la plante ne peut pas tolérer la période d'incubation, mais cet obstacle peut être surmonté en obligeant les ovipares à pondre leurs oeufs sur de pousses de graminées, comme Poa annua, hôte convenable pour les fondatrices. Les ovipares peuvent aussi pondre sans difficultés sur autre chose que des végétaux, et des récipients peuvent ètre mis à incuber sans contenir du matériel végétal.

     

Studies on direct and indirect effects of DNA damage on mutagenesis in monkey cells using an SV40-based shuttle vector

We are using an SV40-based shuttle vector, pZ189, to study mechanisms of mutagenesis in mammalian cells. The vector can be treated with mutagens in vitro and replicated in animal cells; resulting mutants can be selected and amplified in bacteria for DNA sequencing. This versatile vector system has allowed us to explore several different questions relating to the mutagenic process. We have studied the direct effects of template damage caused by UV or benzo[a]pyrene diolepoxide by treating vector DNA with these agents and then replicating the damaged DNA in monkey cells. Mutational mechanisms were deduced from the spectrum of mutations induced in the supF target gene of the vector DNA. To study the role of indirect effects of DNA damage on mutagenesis in mammalian cells, we have treated the cells and the vector DNA separately with DNA-damaging agents. We find that pretreatment of cells with DNA-damaging agents, or with conditioned medium from damaged cells, causes an enhancement of mutagenesis of a UV-damaged vector. Thus, DNA damage can act indirectly to enhance the mutagenic process. We also have preliminary evidence that pZ189 can be used in an in vitro DNA replication system to study the process of mutation fixation on the biochemical level. We believe that the pZ189 vector will prove to be as useful for in vitro studies of mutational mechanisms as it has been for in vivo studies.     

Zeatin Glycosylation Enzymes in Phaseolus: Isolation of O-Glucosyltransferase from P. lunatus and Comparison to O-Xylosyltransferase from P. vulgaris

An enzyme catalyzing the formation of O-glucosylzeatin in immature embryos of Phaseolus lunatus was purified 2500-fold using ammonium sulfate precipitation followed by affinity and anion exchange chromatography. The enzyme uses trans-zeatin as substrate (K_m 28 micromolar) but not cis-zeatin, ribosylzeatin, or dihydrozeatin. Both UDP-glucose and UDP-xylose can serve as glycosyl donors, with K_ms of 0.2 and 2.7 millimolar, respectively, for the formation of O-glucosylzeatin and O-xylosylzeatin. In comparison, the UDPxylose-zeatin:O-xylosyltransferase (JE Turner, DWS Mok, MC Mok, G Shaw [1987] Proc Natl Acad Sci USA 84: 3714-3717) isolated by the same procedures from P. vulgaris embryos uses only UDP-xylose as donor substrate and the K_ms for both zeatin and UDP-xylose are much lower (2 and 3 micromolar, respectively). The chromatographic behavior on affinity columns and molecular weights (approximate M_r 44,000 daltons) of the two enzymes are similar. Results from substrate competition experiments and enzyme separation by anion exchange HPLC indicate a single, distinct, zeatin O-glycosylation enzyme occurs in embryos of each of these Phaseolus species.     

Infection with the human T-lymphotropic virus type I. A review for clinicians.

The human T-cell lymphotropic virus type I (HTLV-I) is the first retrovirus identified in humans. It has been responsible for a number of clinical syndromes, most notably adult T-cell leukemia or lymphoma and tropical spastic paraparesis. In the United States, infection with this virus is most frequently found in specific subsets of our population, particularly in those who live in the southeastern states, have southern Japanese ancestry, or share intravenous drug paraphernalia. Understanding the epidemiology and clinical manifestations of this virus is necessary to properly diagnose and care for patients with HTLV-I infection.